Factor VIIa Interaction with Endothelial Cell Protein C Receptor

因子 VIIa 与内皮细胞蛋白 C 受体的相互作用

基本信息

批准号：
8403678
负责人：
Vijaya Mohan Rao Lella
金额：
$ 33.56万
依托单位：
UNIVERSITY OF TEXAS HLTH CTR AT TYLER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8403678
关键词：
Address Affinity Animal Model Antibodies Anticoagulants Antigens Binding Binding Sites Biological Models Blood Blood Circulation Blood Coagulation Factor Blood Coagulation Factor VII Blood Vessels Caveolae Cell Line Cell model Clinical Data Disease Dose Dynamin Endocytosis Endothelial Cells Endothelium Extravasation Factor VIIa Fluorescence Resonance Energy Transfer Functional disorder Future Generations Hemophilia A Hemorrhage Hemostatic Agents Hemostatic function Immunohistochemistry In Vitro Inflammation Inflammatory Joints Knockout Mice Knowledge Lead Ligands Measures Mediating Microscopy Modus Molecular Mus PAR-1 Receptor Pathway interactions Patients Physiological Play Positioning Attribute Protein C Proteins Publishing Receptor Activation Receptor Signaling Recombinants Recycling Regulation Role Sepsis Signal Pathway Signal Transduction Surface System TFPI Testing Therapeutic Thrombin Thrombomodulin Thromboplastin Tissues Transmission Electron Microscopy Treatment Cost Treatment Efficacy Withholding Treatment activated Protein C activated protein C receptor base bone caveolin 1 cofactor improved in vivo in vivo Model inhibitor/antagonist joint destruction mouse model novel novel therapeutics overexpression prophylactic receptor receptor mediated endocytosis recombinant FVIIa transcytosis treatment strategy

项目摘要

PROJECT SUMMARY/ABSTRACT The endothelial cell lining constitutes the interface between vascular tissue and circulating blood, and plays an important role in regulation of molecular exchange between blood and perivascular tissues. Although endothelial cells are positioned optimally to interact with circulating clotting factors, very little is known about these interactions. Recently, we have shown that factor VIIa (FVIIa) binds specifically to endothelial cell protein C receptor (EPCR) on endothelial cells. Both FVII and FVIIa bind to EPCR in a true-ligand fashion and with a similar affinity as that of protein C and activated protein C (APC) to EPCR. At present the physiological significance and importance of FVII/FVIIa interaction with EPCR are unknown. Our recent studies showed that FVIIa binding to EPCR resulted in its endocytosis. These studies also indicate that EPCR-mediated endocytosis and recycling may facilitate the transport of FVIIa from the luminal to abluminal surface. Our latest studies show that FVIIa binding to EPCR on endothelial cells activates protease activated receptor-1 (PAR1) and provides endothelial barrier protection. Studies in cell model systems have suggested that therapeutic concentrations of FVIIa may down-regulate the protein C anticoagulant pathway by displacing protein C from EPCR on the endothelium. All of these findings are novel and cumulatively indicate that FVIIa interaction with EPCR may play an important role in pathophysiology. Based on these findings, we hypothesize that FVIIa interaction with EPCR on the endothelium modulates FVIIa transport, provides protection against vascular leakage, and down-regulates the protein C/APC anticoagulant pathway in therapeutical conditions. Three specific aims that test these hypotheses are, (1) investigate the role of EPCR in FVIIa transcytosis and clearance from bloodstream, (2) elucidate EPCR-FVIIa-mediated barrier protective mechanism(s) in endothelial cells and define the role of FVIIa-EPCR signaling in maintaining vascular barrier integrity in vivo, and (3) test the hypothesis that pharmacological concentrations of rFVIIa down-regulate the protein C/APC-mediated anticoagulant pathway and thereby potentiate the hemostatic effect of rFVIIa in the treatment of bleeding disorders. For these studies, we will use both cell and animal model systems. We will employ well characterized EPCR deficient and EPCR overexpressing mice and antibodies specific to mouse EPCR in order to investigate the role of EPCR in FVIIa transport and mediating FVIIa-induced cellular effects. FVIIa interaction with the endothelium and its transport will be probed by immunohistochemistry, transmission electron microscopy and measuring FVII/FVIIa activity and antigen in tissues. Confocal and fluorescence resonance energy transfer (FRET) microscopy will be used to investigate the physical interaction of FVIIa, EPCR and PAR1. The proposed studies will reveal novel functions of EPCR and FVIIa and revise our current understanding on the role of FVIIa and EPCR in hemostasis and inflammation. The proposed studies will have major and important implications for future treatment strategies of bleeding disorders and sepsis.

项目概要/摘要内皮细胞衬里构成血管组织和循环血液之间的界面，并发挥着重要作用在调节血液和血管周围组织之间的分子交换中发挥重要作用。虽然内皮细胞处于最佳位置以与循环凝血因子相互作用，但对此知之甚少这些相互作用。最近，我们发现因子 VIIa (FVIIa) 与内皮细胞蛋白特异性结合内皮细胞上的 C 受体 (EPCR)。 FVII 和 FVIIa 均以真正的配体方式与 EPCR 结合，并且具有与蛋白 C 和活化蛋白 C (APC) 对 EPCR 的亲和力相似。目前生理 FVII/FVIIa 与 EPCR 相互作用的意义和重要性尚不清楚。我们最近的研究表明 FVIIa 与 EPCR 结合导致其内吞作用。这些研究还表明，EPCR 介导的内吞作用和再循环可能促进 FVIIa 从腔表面转运至腔外表面。我们最新的研究表明，FVIIa 与内皮细胞上的 EPCR 结合可激活蛋白酶激活受体 1 (PAR1) 并提供内皮屏障保护。细胞模型系统的研究表明，治疗 FVIIa 浓度可能通过取代蛋白 C 来下调蛋白 C 抗凝途径内皮上的 EPCR。所有这些发现都是新颖的，并且累积表明 FVIIa 与 EPCR可能在病理生理学中发挥重要作用。基于这些发现，我们假设 FVIIa 与内皮上的 EPCR 相互作用，调节 FVIIa 转运，提供针对血管的保护渗漏，并在治疗条件下下调蛋白 C/APC 抗凝途径。三检验这些假设的具体目标是，(1) 研究 EPCR 在 FVIIa 转胞吞作用中的作用，以及从血流中清除，(2) 阐明 EPCR-FVIIa 介导的屏障保护机制内皮细胞并定义 FVIIa-EPCR 信号在维持血管屏障完整性中的作用体内，并且（3）检验 rFVIIa 的药理学浓度下调的假设蛋白 C/APC 介导的抗凝途径，从而增强 rFVIIa 的止血作用用于治疗出血性疾病。对于这些研究，我们将使用细胞和动物模型系统。我们将采用特征明确的 EPCR 缺陷型和 EPCR 过表达型小鼠以及针对 EPCR 的特异性抗体。小鼠 EPCR，以研究 EPCR 在 FVIIa 转运和介导 FVIIa 诱导的细胞中的作用影响。 FVIIa 与内皮细胞的相互作用及其转运将通过免疫组织化学进行探测，透射电子显微镜并测量组织中的 FVII/FVIIa 活性和抗原。共焦和荧光共振能量转移（FRET）显微镜将用于研究物理相互作用 FVIIa、EPCR 和 PAR1。拟议的研究将揭示 EPCR 和 FVIIa 的新功能，并修订我们的研究目前对 FVIIa 和 EPCR 在止血和炎症中的作用的了解。拟议的研究将对出血性疾病和败血症的未来治疗策略产生重大影响。